1. Field of the Invention
The present invention relates to an enzyme N-acetyl-D-glucosamine deacetylase capable of hydrolyzing the acetamide group of N-acetyl-D-glucosamine to produce D-glucosamine and acetic acid. More particularly, the present invention relates to a deacetylase which acts specifically on the monomer of N-acetyl-D-glucosamine, and not on the oligomers including dimer and the polymer.
The present invention also relates to a process for preparing the above described enzyme, and to a process for hydrolyzing said acetamido group using the deacetylase of the present invention.
2. Description of the Prior Art
D-glucosamine, the deacetylated product of N-acetyl-D-glucosamine, is useful as pharmaceuticals, foodstuffs, and intermediates for the preparation of pharmaceuticals. In addition, oligosaccharides consisting of D-glucosamine unit have increasingly been drawing attention and becoming important as having valuable physiological activities such as antibacterial, antifungal and antitumor activities.
D-glucosamine has hitherto been prepared from N-acetyl-D-glucosamine which is prepared from purified chitin (.beta.-1,4-poly-N-acetyl-D-glucosamine, obtained from the cuticle (crust) of a crustacean such as prawn or crab by removing inorganic salts, proteins and lipids), by deacetylating with a conc. alkali or mineral acid.
However, the hydrolysis of the N-acetyl group of N-acetyl-D-glucosamine does not progress so easily and therefore, relatively hard reaction conditions are necessary to complete the hydrolysis, for example, heating for a long period of time in a concentrated acid or alkali such as 30-60% solution, leading to an increase in the formation of undesirable by-products, and the yield of the desired D-glucosamine is low. Moreover, the elimination of the acid or alkali used necessitates additional elaboration and expense.
There have been a need for developping more mild methods of deacetylating N-acetyl-D-glucosamine in a high yield without the formation of undesirable by-products, and a biological method using an enzyme capable of deacetylating N-acetyl-D-glucosamine has been investigated.
As biological deacetylation methods, there have been several references known wherein microorganisms belonging to Mucor sp, Aeromonas sp. or Colletotrium sp. are used.
In a method using a microorganism belonging to Mucor sp., soluble glycol chitin and the pentamer are deacetylated to the extent of 12-14%, but there is no disclosure of a deacetylase acting only on the monomer of N-acetyl-D-glucosamine [Y. Araki, E. Ito, Biochem. Biophys. Res. Commun., 56: 669 (1974); Eur. J. Biochem., 55:71 (1975); Methods Enzymol., 161:510 (1988); L. L. Davis, S. Bartnicki-Garcia, Biochemistry, 23: 1065 (1984); C. Calvo-Mendez, J. Ruiz-Herrera, Exp. Mycol., 1:128 (1987)].
A method using a microorganism belonging to Aeromonas sp. intends to deacetylate solid chitin of high polymerization degree, and there is no disclosure of deacetylating monosaccharide [K. Shimahara, H. Iwasaki, Asahi Garasu Kogyo Gijutsu Shoreikai, 41: 299 (1982), C.A. 99:84876v (1983)].
There is no disclosure also of deacetylating monosaccharide using a microorganism belonging to Colletotrium sp. [H. Kauss, W. Jeblick, D. H. Young, Plant Sci. Lett. 28: 231 (1983); H. Kauss, B. Bauch, Methods in Enzymology, 161: 518 (1988)].
It has now surprisingly been found that a marine microorganism belonging to Vibrio sp. produces an enzyme which is capable of splitting the acetyl group of N-acetyl-D-glucosamine and which specifically acts on the monomer of N-acetyl-D-glucosamine and not on the oligomer or polymer thereof.
The enzyme as described above having such novel substrate specificity has never been known hitherto, and represents a useful functional enzyme.